Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate

Serine proteases belong to the class of hydrolytic enzymes that cleave amide or ester bonds. Three amino acids, a serine, a histidine, and an aspartic acid, located at rather distant positions in the sequence, are folded in characteristic proximity to one another. The carbonyl carbon of the scissile peptide bond is nucleophilically attacked by the hydroxyl oxygen of serine. The nucleophilicity of the OH group is enhanced by an H-bond to an adjacent imidazole group of a histidine, which accepts a proton from the OH group and is thereby transferred to a positively charged state. The adjacent aspartate residue compensates for the positive charge. The simultaneously generated negative charge on the former carbonyl oxygen is stabilized by NH functions in the H-bond-donating oxyanion hole. The N-terminal portion of the peptide substrate is released, while the C-terminal portion remains covalently bound as an acyl enzyme complex. The residues involved in substrate binding can vary, in particular, the nucleophilic serine can be replaced by a threonine or a cysteine, resulting in threonine and cysteine proteases. Small binding pockets on the protease surface, which accommodate the amino acid side chains at the C-terminal end adjacent to the cleavage site, primarily recognize the peptide chain to be cleaved. The composition of the pockets determines the chemical building blocks required for inhibitor design to develop highly potent ligands. A number of warhead groups are known to either reversibly or irreversibly block the catalytic serine, threonine or cysteine residue. Blood coagulation is a highly regulated cascade of serine proteases. Effective inhibitors for antithrombotic therapy have been developed for thrombin and factor Xa, which are involved in the final steps of the cascade. Irreversible inhibition by covalent bond formation with the catalytic serine is used to block lipases or transpeptidases. Covalent binding is achieved with a drug that has a reactive, highly strained lactone or lactam ring that opens upon binding (penicillins and cephalosporins). A stable acyl form of the enzyme is formed that does not allow further conversions in the catalytic center. The β-lactamases, which are structurally closely related to the transpeptidases, hydrolyze the acyl form of the enzyme. They orient a polar glutamate residue into the catalytic center, thereby stabilizing a water molecule in an optimal position for nucleophilic attack. In transpeptidases, this site is inaccessible to water due to hydrophobic residues. Lactamase inhibitors break this resistance by blocking the water position with a polar side chain. Many cysteine proteases are found in bacteria, parasites and viruses. By replacing the OH group with an SH group, the transition state in cysteine proteases appears to be closer to the acyl enzyme form and the thiol group is thought to be in a deprotonated state. The strategy to inhibit these enzymes is usually a head group that covalently attaches to the sulfur. The first orally available SARS-CoV-2 cysteine protease inhibitor has been launched to market. Transglutaminases follow a very similar enzymatic mechanism to cysteine proteases. However, instead of cleaving a peptide bond in the main chain, they form an isopeptide bond between the terminal amino group of a lysine and the carboxylate group of a glutamate. https://sn.pub/gqj47j